Michel van Weeghel

Riboflavin-responsive oxidative phosphorylation complex I deficiency caused by defective ACAD9: new function for an old gene

Mitochondrial complex I deficiency is the most common oxidative phosphorylation defect. Mutations have been detected in mitochondrial and nuclear genes, but the genetics of many patients remain unresolved and new genes are probably involved. In a consanguineous family, patients presented easy fatigability, exercise intolerance and lactic acidosis in blood from early childhood. In muscle, subsarcolemmal mitochondrial proliferation and a severe complex I deficiency were observed. Exercise intolerance and complex I activity was improved by a supplement of riboflavin at high dosage. Homozygosity mapping revealed a candidate region on chromosome three containing six mitochondria-related genes. Four genes were screened for mutations and a homozygous substitution was identified in ACAD9 (c.1594 C>T), changing the highly conserved arginine-532 into tryptophan. This mutation was absent in 188 ethnically matched controls. Protein modelling suggested a functional effect due to the loss of a stabilizing hydrogen bond in an α-helix and a local flexibility change. To test whether the ACAD9 mutation caused the complex I deficiency, we transduced fibroblasts of patients with wild-type and mutant ACAD9. Wild-type, but not mutant, ACAD9 restored complex I activity. An unrelated patient with the same phenotype was compound heterozygous for c.380 G>A and c.1405 C>T, changing arginine-127 into glutamine and arginine-469 into tryptophan, respectively. These amino acids were highly conserved and the substitutions were not present in controls, making them very probably pathogenic. Our data support a new function for ACAD9 in complex I function, making this gene an important new candidate for patients with complex I deficiency, which could be improved by riboflavin treatment.

Post-natal myogenic and adipogenic developmental:Defects and metabolic impairment upon loss of a-type lamins

A-type lamins are a major component of the nuclear lamina. Mutations in the LMNA gene, which encodes the A-type lamins A and C, cause a set of phenotypically diverse diseases collectively called laminopathies. While adult LMNA null mice show various symptoms typically associated with laminopathies, the effect of loss of lamin A/C on early post-natal development is poorly understood. Here we developed a novel LMNA null mouse (LMNAGT-/-) based on genetrap technology and analyzed its early post-natal development. We detect LMNA transcripts in heart, the outflow tract, dorsal aorta, liver and somites during early embryonic development. Loss of A-type lamins results in severe growth retardation and developmental defects of the heart, including impaired myocyte hypertrophy, skeletal muscle hypotrophy, decreased amounts of subcutaneous adipose tissue and impaired ex vivo adipogenic differentiation. These defects cause death at 2 to 3 weeks post partum associated with muscle weakness and metabolic complications, but without the occurrence of dilated cardiomyopathy or an obvious progeroid phenotype. Our results indicate that defective early post-natal development critically contributes to the disease phenotypes in adult laminopathies.

Mitochondrial long chain fatty acid β-oxidation in man and mouse

Several mouse models for mitochondrial fatty acid beta-oxidation (FAO) defects have been developed. So far, these models have contributed little to our current understanding of the pathophysiology. The objective of this study was to explore differences between murine and human FAO. Using a combination of analytical, biochemical and molecular methods, we compared fibroblasts of long chain acyl-CoA dehydrogenase knockout (LCAD(-/-)), very long chain acyl-CoA dehydrogenase knockout (VLCAD(-/-)) and wild type mice with fibroblasts of VLCAD-deficient patients and human controls. We show that in mice, LCAD and VLCAD have overlapping and distinct roles in FAO. The absence of VLCAD is apparently fully compensated, whereas LCAD deficiency is not. LCAD plays an essential role in the oxidation of unsaturated fatty acids such as oleic acid, but seems redundant in the oxidation of saturated fatty acids. In strong contrast, LCAD is neither detectable at the mRNA level nor at the protein level in men, making VLCAD indispensable in FAO. Our findings open new avenues to employ the existing mouse models to study the pathophysiology of human FAO defects.

Fasting-induced myocardial lipid accumulation in long-chain acyl-CoA dehydrogenase knockout mice is accompanied by impaired left ventricular function

Background— Lipotoxicity may be a key contributor to the pathogenesis of cardiac abnormalities in mitochondrial long-chain fatty acid &bgr;-oxidation (FAO) disorders. Few data are available on myocardial lipid levels and cardiac performance in FAO deficiencies. The purpose of this animal study is to assess fasting-induced changes in cardiac morphology, function, and triglyceride (TG) storage as a consequence of FAO deficiency in a noninvasive fashion. Methods and Results— MRI and proton magnetic resonance spectroscopy (1H-MRS) were applied in vivo in long-chain acyl-CoA dehydrogenase (LCAD) knockout (KO) mice and wild-type (WT) mice (n=8 per genotype). Fasting was used to increase the hearts dependency on FAO for maintenance of energy homeostasis. In vivo data were complemented with ex vivo measurements of myocardial lipids. Left ventricular (LV) mass was higher in LCAD KO mice compared with WT mice (P<0.05), indicating LV myocardial hypertrophy. Myocardial TG content was higher in LCAD KO mice at baseline (P<0.001) and further increased in fasted LCAD KO mice (P<0.05). Concomitantly, LV ejection fraction (P<0.01) and diastolic filling rate (P<0.01) decreased after fasting, whereas these functional parameters did not change in fasted WT mice. Myocardial ceramide content was higher in fasted LCAD KO mice compared with fasted WT mice (P<0.05). Conclusions— Using a noninvasive approach, this study reveals accumulation of myocardial TG in LCAD KO mice. Toxicity of accumulating lipid metabolites such as ceramides may be responsible for the fasting-induced impairment of cardiac function observed in the LCAD KO mouse.

Functional redundancy of mitochondrial enoyl-CoA isomerases in the oxidation of unsaturated fatty acids

Mitochondrial enoyl‐CoA isomerase (ECI1) is an auxiliary enzyme involved in unsaturated fatty acid oxidation. In contrast to most of the other enzymes involved in fatty acid oxidation, a deficiency of ECI1 has yet to be identified in humans. We used wild‐type (WT) and Eci1‐deficient knockout (KO) mice to explore a potential presentation of human ECI1 deficiency. Upon food withdrawal, Eci1‐deficient mice displayed normal blood β‐hydroxybutyrate levels (WT 1.09 mM vs. KO 1.10 mM), a trend to lower blood glucose levels (WT 4.58 mM vs. KO 3.87 mM, P=0.09) and elevated blood levels of unsaturated acylcarnitines, in particular C12:1 acylcarnitine (WT 0.03 μM vs. KO 0.09 μM, P<0.01). Feeding an olive oil‐rich diet induced an even greater increase in C12:1 acylcarnitine levels (WT 0.01 μM vs. KO 0.04 μM, P<0.01). Overall, the phenotypic presentation of Eci1‐deficient mice is mild, possibly caused by the presence of a second enoyl‐CoA isomerase (Eci2) in mitochondria. Knockdown of Eci2 in Eci1‐deficient fibroblasts caused a more pronounced accumulation of C12:1 acylcarnitine on incubation with unsaturated fatty acids (12‐fold, P<0.05). We conclude that Eci2 compensates for Eci1 deficiency explaining the mild phenotype of Eci1‐deficient mice. Hypoglycemia and accumulation of C12:1 acylcarnitine might be diagnostic markers to identify ECI1 deficiency in humans.—van Weeghel, M., te Brinke, H., van Lenthe, H., Kulik, W., Minkler, P. E., Stoll, M. S. K., Sass, J. O., Janssen, U., Stoffel, W., Schwab, O. K., Wanders, R. J. A., Hoppel, C. L., Houten, S. M. Functional redundancy of mitochondrial enoyl‐CoA isomerases in the oxidation of unsaturated fatty acids. FASEB J. 26, 4316–4326 (2012). www.fasebj.org

Role of medium- and short-chain L-3-hydroxyacyl-CoA dehydrogenase in the regulation of body weight and thermogenesis.

Dysregulation of fatty acid oxidation plays a pivotal role in the pathophysiology of obesity and insulin resistance. Medium- and short-chain-3-hydroxyacyl-coenzyme A (CoA) dehydrogenase (SCHAD) (gene name, hadh) catalyze the third reaction of the mitochondrial β-oxidation cascade, the oxidation of 3-hydroxyacyl-CoA to 3-ketoacyl-CoA, for medium- and short-chain fatty acids. We identified hadh as a putative obesity gene by comparison of two genome-wide scans, a quantitative trait locus analysis previously performed in the polygenic obese New Zealand obese mouse and an earlier described small interfering RNA-mediated mutagenesis in Caenorhabditis elegans. In the present study, we show that mice lacking SCHAD (hadh−/−) displayed a lower body weight and a reduced fat mass in comparison with hadh+/+ mice under high-fat diet conditions, presumably due to an impaired fuel efficiency, the loss of acylcarnitines via the urine, and increased body temperature. Food intake, total energy expenditure, and locomotor activity were not altered in knockout mice. Hadh−/− mice exhibited normal fat tolerance at 20 C. However, during cold exposure, knockout mice were unable to clear triglycerides from the plasma and to maintain their normal body temperature, indicating that SCHAD plays an important role in adaptive thermogenesis. Blood glucose concentrations in the fasted and postprandial state were significantly lower in hadh−/− mice, whereas insulin levels were elevated. Accordingly, insulin secretion in response to glucose and glucose plus palmitate was elevated in isolated islets of knockout mice. Therefore, our data indicate that SCHAD is involved in thermogenesis, in the maintenance of body weight, and in the regulation of nutrient-stimulated insulin secretion.

Food withdrawal lowers energy expenditure and induces inactivity in long-chain fatty acid oxidation–deficient mouse models

Very long‐chain acyl‐CoA dehydrogenase (VLCAD) deficiency is an inherited disorder of mitochondrial long‐chain fatty acid β‐oxidation (FAO). Patients with VLCAD deficiency may present with hypoglycemia, hepatomegaly, cardiomyopathy, and myopathy. Although several mouse models have been developed to aid in the study of the pathogenesis of long‐chain FAO defects, the muscular phenotype is underexposed. To address the muscular phenotype, we used a newly developed mouse model on a mixed genetic background with a more severe defect in FAO (LCAD–/–; VLCAD+/–) in addition to a validated mouse model (LCAD–/–; VLCAD+/+) and compared them with wild‐type (WT) mice. We found that both mouse models show a 20% reduction in energy expenditure (EE) and a 3‐fold decrease in locomotor activity in the unfed state. In addition, we found a 1.7°C drop in body temperature in unfed LCAD–/–; VLCAD+/+ mice compared with WT body temperature. We conclude that food withdrawal‐induced inactivity, hypothermia, and reduction in EE are novel phenotypes associated with FAO deficiency in mice. Unexpectedly, inactivity was not explained by rhabdomyolysis, but rather reflected the overall reduced capacity of these mice to generate heat. We suggest that mice are partly protected against the negative consequence of an FAO defect.—Diekman, E. F., van Weeghel, M., Wanders, R. J. A., Visser, G., Houten, S. M. Food withdrawal lowers energy expenditure and induces inactivity in long‐chain fatty acid oxidation–deficient mouse models. FASEB J. 28, 2891–2900 (2014). www.fasebj.org

Identification and characterization of Eci3, a murine kidney-specific Δ3,Δ2-enoyl-CoA isomerase

Oxidation of unsaturated fatty acids requires the action of auxiliary enzymes, such as Δ3, Δ2‐enoyl‐CoA isomerases. Here we describe a detailed biochemical, molecular, histological, and evolutionary characterization of Eci3, the fourth member of the mammalian enoyl‐CoA isomerase family. Eci3 specifically evolved in rodents after gene duplication of Eci2. Eci3 is with 79% identity homologous to Eci2 and contains a peroxisomal targeting signal type 1. Subcellular fractionation of mouse kidney and immunofluorescence studies revealed a specific peroxisomal localization for Eci3. Expression studies showed that mouse Eci3 is almost exclusively expressed in kidney. By using immunohistochemistry, we found that Eci3 is not only expressed in cells of the proximal tubule, but also in a subset of cells in the tubulointerstitium and the glomerulus. In vitro, Eci3 catalyzed the isomerization of trans‐3‐nonenoyl‐CoA to trans‐2‐nonenoyl‐CoA equally efficient as Eci2, suggesting a role in oxidation of unsaturated fatty acids. However, in contrast to Eci2, in silico gene coexpression and enrichment analysis for Eci3 in kidney did not yield carboxylic acid metabolism, but diverse biological functions, such as ion transport (P=7.1E‐3) and tissue morphogenesis (P= 1.0E‐3). Thus, Eci3 picked up a novel and unexpected role in kidney function during rodent evolution.—van Weeghel, M., Ofman, R., Argmann, C. A., Ruiter, J. P. N., Claessen, N., Oussoren, S. V., Wanders, R. J. A., Aten, J., Houten, S. M. Identification and characterization of Eci3, a murine kidney‐specific Δ3, Δ2‐enoyl‐CoA isomerase. FASEB J. 28, 1365–1374 (2014). www.fasebj.org

WormJam: A consensus C. elegans Metabolic Reconstruction and Metabolomics Community and Workshop Series

Janna Hastings, Abraham Mains, Marta Artal-Sanz, Sven Bergmann, Bart P. Braeckman, Jake Bundy , Filipe Cabreiro, Paul Dobson, Paul Ebert, Jake Hattwell, Hooman Hefzi, Riekelt H. Houtkooper, Rob Jelier, Chintan Joshi, Varun B. Kothamachu, Nathan Lewis, Artur Bastos Lourenço, Yu Nie, Povilas Norvaisas, Juliette Pearce, Cristian Riccio, Nicolas Rodriguez, Toon Santermans, Pasquale Scarcia, Horst Joachim Schirra, Ming Sheng, Reuben Smith, Manusnan Suriyalaksh, Benjamin Towbin, Mary Ann Tuli, Michel van Weeghel, David Weinkove, Aleksandra Ze ci c, Johannes Zimmermann, Nicolas le Nov ere, Christoph Kaleta, Michael Witting, and Olivia Casanueva Epigenetics, Babraham Institute, Babraham Research Campus, Cambridge, UK; Developmental Biology, Andalusian Center for Developmental Biology. Consejo Superior de Investigaciones Cient ıficas/Junta de Andalucia/Universidad Pablo de Olavide, Seville, Spain; Computational Biology, University of Lausanne, Lausanne, Switzerland; Department of Biology, University of Gent, Gent, Belgium; Computational and Systems Medicine, Imperial College London, London, UK; Structural and Molecular Biology, University College London, London, UK; School of Computer Science, University of Manchester, Manchester, UK; School of Biological Sciences, University of Queensland, Queensland, Australia; Centre for Advanced Imaging, University of Queensland, Queensland, Australia; Department of Bioengineering, Novo Nordisk Center for Biosustainability at UC San Diego, University of California, San Diego, USA; Laboratory Genetic Metabolic Diseases, Academic Medical Center, Amsterdam, Netherlands; Centre of Microbial and Plant Genetics, KU Leuven, Leuven, Belgium; Department of Pediatrics, Novo Nordisk Center for Biosustainability at UC San Diego, University of California, San Diego, USA; Signalling, Babraham Institute, Babraham Research Campus, Cambridge, UK; School of Human and Life Sciences, Canterbury Christ Church University, Canterbury, UK; Sanger Institute, University of Cambridge, Cambridge, UK; Dep. Biosciences Biotechnologies Biopharmaceutics, University of Bari, Bari, Italy; Neurobiology, MRC LMB, Cambridge, UK; Friedrich Miescher Institute, Basel, Switzerland; WormBase, Caltech, Pasadena, CA, USA; Department of Biosciences, Durham University, Durham, UK; Medical Systems Biology, Christian-Albrechts-University Kiel, Kiel, Germany; Research Unit Analytical BioGeoChemistry, Helmholtz Zentrum Muenchen, Muenchen, Germany

Glycine promotes longevity in Caenorhabditis elegans in a methionine cycle-dependent fashion

The deregulation of metabolism is a hallmark of aging. As such, changes in the expression of metabolic genes and the profiles of amino acid levels are features associated with aging animals. We previously reported that the levels of most amino acids decline with age in Caenorhabditis elegans (C. elegans). Glycine, in contrast, substantially accumulates in aging C. elegans. In this study we show that this is coupled to a decrease in gene expression of enzymes important for glycine catabolism. We further show that supplementation of glycine significantly prolongs C. elegans lifespan and ameliorates specific transcriptional changes that are associated with aging. Glycine feeds into the methionine cycle. We find that mutations in components of this cycle, methionine synthase (metr-1) and S-adenosylmethionine synthetase (sams-1), completely abrogate glycine-induced lifespan extension. Strikingly, the beneficial effects of glycine supplementation are conserved when we supplement with serine, also driving the methionine cycle. RNA sequencing of serine- and glycine-supplemented worms reveals similar transcriptional profiles including widespread gene suppression. Taken together, these data uncover a novel role of glycine in the deceleration of aging through its function in the methionine cycle. Author summary There are a growing number of studies showing that amino acids function as signal metabolites that influence aging and health. Although contemporary -OMICs studies have uncovered various associations between metabolite levels and aging, in many cases the directionality of the relationships is unclear. In a recent metabolomics study, we found that glycine accumulates in aged C. elegans while other amino acids decrease. The present study shows that glycine supplementation prolongs longevity and drives a genome-wide inhibition effect on C. elegans gene expression. Glycine as a one-carbon donor fuels the methyl pool of one-carbon metabolism composed of folates and methionine cycle. We find that glycine-mediated longevity effect is fully dependent on methionine cycle, and that all of these observations are conserved with supplementation of the other one-carbon amino acid, serine. These results provide a novel role for glycine as a promoter of longevity and bring new insight into the role of one-carbon amino acids in the regulation of aging that may ultimately be beneficial for humans.